Cells are highly complex entities and their study requires the collection of information on multiple interacting components and thus multiple parameters (multiplexing). The conventional approach is to use multiple fluorescent species introduced into (e.g. FITC) or synthesised within (e.g. GFP) or naturally occurring in (e.g. NADH) the sample, and matching detection channels (for different emission wavelengths) to collect such information, one channel per label. This approach is generally limited to about three or four (N==3 or 4) multiplexed fluorescent species due to the spectral emission overlap of the labels and the spectral discriminating ability of the detector under low light conditions. Alternatively, fluorescent species may be distinguished on the basis of their fluorescent lifetime. Again, this is limited to a modest number of detection channels at any one time (M=2 or 3). Conventionally these detection modalities are available in different instruments and the information obtained must be correlated between experiments. Furthermore, the measurements have typically been made in cuvettes, which may contain complex mixtures of several components.
Spectrally Resolved Fluorescence
Fluorescent species (fluorophores) typically exhibit an excitation spectrum (in the form of a peak) within a shorter wavelength range, and an emission spectrum (in the form of a peak) with a longer wavelength range (FIG. 1a), whereby the excitation spectrum describes the probability of an incident photon exciting the species in a ground state according to its wavelength. After a short period, the excited species typically emits a photon to return to the ground state. The probability that the emitted photon has a particular wavelength is described by the emission spectrum.
The fluorescence process is inefficient and the emission light is much less intense than the excitation light employed, typically much less than 1%. In a practical embodiment, a particular species is typically excited by light from a source with light emitted across a particular excitation waveband. Of the emitted light, a particular emission or detection waveband may be selected for delivery to a detection subsystem (FIG. 1b). The excitation may be a narrow band source such as a laser, or a broader band source such as a lamp (e.g. Xenon or Mercury) from which a suitable waveband is selected. The emission waveband may be selected by the use of an optical filter.
Since there is typically an overlap between the excitation spectrum and the emission spectrum of the fluorescent species, this places practical constraints on the excitation waveband and emission or detection wavebands that may be employed. It is desirable to employ a broad emission or detection waveband, since this gives a greater probability that emitted light is detected, and the more sensitive the instrument may be to that species. Since the excitation light is typically much more intense than the emission light, it is usually necessary to ensure that the emission waveband of the light available to the detector does not include any part of the excitation waveband used which would otherwise overwhelm the detector or cause a spurious background signal in the absence of any fluorescent species. Maintaining a low background is particularly important when the fluorescent species of interest may be present in low concentrations. Therefore, extending the emission or detection waveband towards to shorter wavelengths in order to increase the detection efficiency requires the excitation waveband to be placed further towards the shorter wavelengths, and possible away from the excitation peak, resulting in poorer excitation efficiency. The design of a particular embodiment typically involves a careful tradeoff of selection of excitation waveband and emission or detection wavebands.
When a sample includes two or more fluorescent species, the situation becomes more complex, and there is a further trade off to be made. Either the individual fluorescent species must be interrogated sequentially in time one after another (temporal multiplexing), or the multiple excitation waveband and emission wavebands must be selected so as to avoid interference. There is likely to be a lost of sensitivity compared detection with a single fluorescent species, since the choice of wavebands width and location is more limited (FIG. 1c). In any case, the fluorescent species employed must be carefully selected, since fluorescent species which have excitation and emission spectra that are too similar may not be distinguishable. Furthermore some fluorescent species have significant secondary peaks in their excitation and emission spectra which causes interference in the detection of multiple species, limiting the available sensitivity and the dynamic range.
Fluorescence detection methods may be applied to samples in cuvettes or wells and are available in a number of commercial instruments marketed by PerkinElmer, Thermo Electron Varioskan and Tecan, for example. With appropriate selection of additional components, they may also be applied as an imaging modality thus creating an image of a sample such as a cell or tissue where each pixel is represented by a number that corresponds to the value measured for that point. Suitable fluorescent microscopes are available from suppliers such as Zeiss, Nikon, Olympus and Leica and suitable components from suppliers such as CRI.
Imaging instruments equipped with multiple spectrally sensitive detectors have been described (32 in the Zeiss 510 META, for example). However, for many fluorescent species with broad emission peaks, the number of photons captured per channel is statistically similar so that it is not possible to overcome the limitation of spectral overlap and sensitivity [Neher 2004]. Typically, an instrument is limited measuring up to 3 fluorescent species when using the visible waveband (400 nm to 700 nm) and 4 fluorescent species if this is extended into the ultraviolet and infrared (250 nm to 1100 nm).
One application area of particular importance in the study of biological systems including research and drug development is a system known of FRET (Fluorescence Resonance Energy Transfer) [Berney 2003]. In this technique a species of interest is labelled with one fluorescent label (the donor) while a second species believed to interact with the first species is labelled with a further fluorescent label (the acceptor) carefully selected so that the emission spectrum of the donor provide a spectral overlap with the excitation spectrum of the acceptor. If the two species of interest come into close proximity (and other factors such as dipole orientation are favourable) then energy may be transferred from an excited donor to the acceptor which can then emit photons. Measuring the change in emission of both the donor and acceptor may then be used to determine the existence or otherwise of FRET and thus if the two species of interest are in close proximity and thus likely to interact. The method is particularly powerful when used in conjunction with biosensor probes where a domain sensitive to a particular species to be sensed is associated with two fluorophores, one acting as a donor and the other as an acceptor, arranged such that the presence of the species to be sensed modifies the geometry of the biosensors such that the configuration of the two fluorophores is altered and FRET is either enhanced or suppressed. Such biosensors sensitive to a number of cellular signalling species have been described, allowing pathways to be studied [Schultz 2005].
However the FRET technique suffers from a number of problems, including difficulty in making reliable measurement using spectral means due to crosstalk in both the excitation and the measurement channels, though these can sometimes be dealt with using adequate controls [Berney 2003]. The substantial spectral bandwidth taken up by the donor and acceptor excitation and emission peaks precludes running more than one FRET system within a given sample.
When the fluorescent species has a well defined excitation and emission spectra, it is possible to achieve a simplification of the situation and to obtain some multiplexing capability. For instance, quantum dots are well known for having a common excitation spectrum and very narrow emission peaks. Thus a single excitation wavelength can be used with several narrow band emission filters to detect several species simultaneously.
Time Resolved Fluorescence
An alternative approach to detecting and distinguishing fluorescent species is to modulate the excitation energy at high speed and to examine the resulting modulation in the emission energy. Many fluorescent species exhibit a characteristic decay (ranging from a few milliseconds to less than a nanosecond) between excitation and emission which is described as the lifetime (time to fall to 1/e of the original intensity). This parameter may be used to distinguish the species.
In FIG. 2 an excitation pulse stimulates species S1 and S2 with characteristic lifetimes T1 and T2, when T1 is much shorter than T2. Typically, this function may be carried out by pulsing the excitation source and timing the arrival of the emitted photons (time correlated single photon counting or TCSPC), measuring the light collected in a given time window after the excitation pulse (or time resolved fluorescence lifetime) or by measuring the phase shift between a frequency modulated source and the detection signal (frequency-domain method). By varying the time window and applying the appropriate corrections, fluorescent species may be distinguished. See [Munster 2005] for a recent review.
Some molecules have quite long lifetimes, with rare earth (Lanthanides) compounds and others (e.g. Ruthenium) exhibiting lifetimes in the range of up to 1000 microseconds and have been used to develop a range of assay methodologies useful in life science research. These are characterised by higher sensitivity and larger dynamic range than those based solely upon spectral discrimination due to the ability of the time resolved property which removed scatter and auto-fluorescence, resulting in better selectivity and contrast.
Many small molecules have lifetimes in the nanosecond range [ISS website]. For example the PURETIME range of molecules have lifetimes in the range 2 ns to 300 ns. Some biological tissues and cellular samples exhibit fluorescence when excited with short wavelength light (500 nm and shorter) due to intrinsic species such as FAD and NADH (auto-fluorescence). These species typically exhibit broadband emission with a short lifetime. This may be used as a source of contrast without additional labelling species, or may interfere with the observation of fluorescent species of interest.
Lifetime detection technologies and applications based on rare earth (Lanthanides) are commonly available as time resolved fluorescence (TRF) and nanosecond lifetime. A number of commercial instruments are available for reading from cuvettes or wells such as PerkinElmer EnVision, Thermo Electron Varioskan, Tecan Ultra, which employ flash lamps or pulsed diode sources. Suitable software converts the collected delay curves into lifetime estimates. Existing systems must be configured for a particular range of lifetime species and cannot deal with more than 2-3 species.
With appropriate selection of additional components, fluorescence lifetime measurement may also be applied as an imaging modality thus creating an image of a sample such as a cell or tissue where each pixel is represented by a number that corresponds to the value measured for that point. A number of such microscopes have been developed, including commercial multi-photon microscopes; customised frequency-domain systems using gated intensifier or multi-channel plate (MCP) with a CCD cameras; and those upgraded with TCSPC systems from suppliers such as Becker and Hickl which again perform the necessary post-processing to compute the lifetime estimates [Munster 2005]. However these systems tend to be very slow, taking many minutes to collect a single image making it hard to monitor kinetic processes.
FRET is a popular application for fluorescence lifetime measurement systems, since the fluorescence lifetime of donor is reduced when in the presence of a suitable acceptor (FIG. 3a). Measuring a change in donor lifetime is often less susceptible to artifacts than the spectral FRET method described earlier. Recently it has been proposed that the use of a ‘dark’ acceptor (one which has a non-radiative decay path and so does not emit and light) [Ganesan 2006] would result in a cleaner system where only the donor emission is observed (FIG. 3b) thus reducing the spectral bandwidth required.
Multiplexing
Existing approaches to discrimination between fluorescent species generally use wavelength only, as shown in FIG. 4a; or lifetime only, as shown in FIG. 4b. In FIG. 4a the two species A and B have a similar lifetime but different emission spectra, so a histogram in the photons in the spectral axis will show two peaks, but only one lifetime; On the other hand in FIG. 4b, A and C have a similar emission spectra but exhibit two distinct lifetimes, so the histogram shown two peaks in the lifetime but only one peak on the wavelength axis.
Limited time and spectrally resolved multiplexing has been described in [Vikström 2004] where two lanthanide (Europium excited at 340 nm, emitting at 615 nm with a characteristic lifetime of 730 us; and Samarium also excited at 340 nm, emitting at 643 nm with a characteristic lifetime 50 us) and one prompt fluorescence label (SYTO24 excited at 485 nm, emitting at 535 nm). However full multiplexing is not described, beyond detecting one species in wavelength, and two in lifetime. This is illustrated in FIG. 4c where F is the small molecule with nanosecond lifetime and 535 nm emission, while D and E are the long lifetime labels (730 us and 50 us both at 615-640 nm). As can be seen, even though there is the potential to discriminate labels with characteristic emission spectra or lifetime alone, the lifetime in the 535 nm channel is not measured.
In a further report [Vikström 2004b] describes an approach to assay 4 readouts: cell stress (using an absorbent dye), cell proliferation (using a Samarium-based label), DNA fragmentation (using a Europium-based label), and cell number (using a fluorescence dye) using a combination of absorbent, time-resolved fluorescence and fluorescence readout modes. The cited advantage is to be able to make all the necessary readings from the same well, thus saving materials and effort in comparison to traditional radioactive readouts. However the readouts are not made at the same time, in particular, the cells are fixed and labelled with antibodies before making the time-resolved fluorescence and fluorescence readout. The approach also uses a well plate reader (the EnVision system from PerkinElmer) with a point (PMT) detector to make the measurements and so does not use any imaging; and does not produce a time course of readings.
In [Hanley 2002] a combined spectral-lifetime microscope is described, this time based on a frequency-domain system with an MCP/CCD detector and a spatial light modulator (SLM) coding system to allow measurements to be made of both spectrum (50 bands across the range 430 nm to 750 nm) and lifetime and reconstructed using a Hadamard transform. The data presented is as shown in FIG. 4d where two populations A and B exhibit different lifetimes across the same emission range; and FIG. 4e with two populations A and B which exhibit variable lifetime according the emission waveband. See also [Hanley 2001].
A further approach to multiplex has been the use of biosensors which are sensitive to more than one biochemical species by changes in the excitation-emission spectrum with the presence of metal ions [Komatsu 2005] or kinases/phospholipases [Schultz 2005]. However these have the drawback that their intensity is both a measure of concentration of the biosensor and also of the species to which it is sensitive, thus confounding the two. Other controls must be put in place if absolute estimates are to be obtained.